Dynamic changes in DNA methylation and hydroxymethylation revealed the transformation of advanced adenoma into colorectal carcinoma

Dear Editor Globally, colorectal carcinoma (CRC) ranks third in terms of prevalence according to the latest Global Cancer Statistics.1 Studies have shown that most CRCs begin as preexisting adenomas,2 among which advanced adenomas (AAs) have been demonstrated to be a more intense risk factor.3 Exploring the underlying mechanism of AACRC transformation is helpful in providing a basis for the precise treatment of CRC. DNA 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) could play major roles in CRC.4,5 A single-tube methylation-specific quantitative polymerase chain reaction (PCR) assay could be a good predictor of CRC recurrence,4 and 5-hmC levels of zw10 kinetochore protein could have a high diagnostic performance for early-stage CRC.5 However, the functions of 5mC and 5hmC in AA-CRC transformation remain unclear. Therefore, we conducted an integrated analysis of 5mC and 5hmC to elucidate the mechanism underlying AA-CRC transformation. Detailed information regarding the study design, participant recruitment andmethodswas provided in Additional File 1. First, to verify the role of 5mC in AA-CRC transformation, 5mC profiles were obtained. Differentially methylated sites (DMSs) and differentially methylated genes (DMGs) were identified (Figure 1A; Figure S1A–C; and Additional File 2). Subsequently, the main biological functions ofDMGswere verified, including ubiquitin-mediated proteolysis, the transforming growth factor-beta pathway, and pluripotency of stem cells (Figure S1D,E; Additional File 3). Based on the characterization of 5mC in AACRC transformation, the 5mC levels in AA and CRC were further investigated. The results showed that 5mC levels were significantly decreased in AA and then markedly increased inCRC, consistentwith the results of sequencing (Figure 1B). Tissue microarrays (TMAs) showed that 5mC levels were also markedly increased in CRC (Figure 1C,D), and patients with CRC with high 5mC levels had a short

overall survival ( Figure 1E). In addition, 5hmC levels were markedly decreased in CRC tissues ( Figure 1F,G). However, no correlation was found between 5hmC levels and the overall survival of patients with CRC ( Figure 1H). The levels of 5mC and 5hmC were further verified using immunofluorescence ( Figure 1I). These results revealed dynamic changes in 5mC and 5hmC during AA-CRC transformation.
5hmC is a stable derivative catalyzed by tet methylcytosine dioxygenases (TETs) in DNA demethylation. To determine dynamic changes in 5mC and 5hmC, we further analyzed 5mC profiles combined with data of 5hmC published in a previous study. 5 Compared with theAA group, the majority of DMSs in 5mC were hypermethylated in the CRC group ( Figure 1A), but the majority of DMSs in 5mC+5hmC were hypermethylated ( Figure 2A). Hypermethylated changes in 5mC mainly occurred in the open sea, and changes in 5mC+5hmC occurred in the open sea, N-shore, S-shore, N-shelf, S-shelf, and CpG island ( Figure 2B). Hypermethylated 5mC DMSs were enriched in other and first exon regions ( Figure 2C), while hypermethylated changes in 5mC+5hmC were enriched in other, the transcriptional start site 1500 and first exon regions ( Figure 2C). In addition, enrichment of hypermethylated changes in 5mC+5hmC in the enhancer was also found ( Figure 2D).
We then performed a combinational analysis of hypermethylated genes and hypohydroxymethylated genes, and 20 overlapped genes were chosen ( Figure 3A). The levels of five genes (ANO10, SUCLG2, PPARGC1A, LRBA, and ATP8A1) were positively correlated with the overall survival of patients with CRC ( Figure S2). Moreover, compared with the AA group, mRNA and protein levels of PPARGC1A, LRBA, and ATP8A1 but not ANO10 and SUCLG2 were both markedly decreased in the CRC group ( Figure 3B-F). The 5hmC levels of PPARGC1A, LRBA, and ATP8A1 were markedly decreased, and the levels of 5mC   were significantly higher in the CRC group than in the AA group ( Figure 3G-I). Analysis of the SurvivalMeth database showed that the 5mC levels of PPARGC1A, LRBA, and ATP8A1 were negatively correlated with the overall survival of patients with CRC ( Figure 3J-L).
To elucidate the mechanism of AA-CRC transformation, the levels of DNA methylases and demethylases were measured. The results showed that DNMT3B levels were significantly increased in AA-CRC transformation but not DNMT1 and DNMT3A ( Figure 4A,B; Figure  S3A-C). TET2 levels were significantly decreased in AA-CRC transformation but not TET1 and TET3 ( Figure 4C,D; Figure S3D-F). Studies have indicated that DNMT3B can accelerate the invasion and migration of CRC and promote CRC development, 6 and TET2 can inhibit CRC progression. 7 Our results showed that DNMT3B knockdown and TET2 overexpression significantly inhibited cell proliferation, invasion, and migration ( Figure 4E-H; Figure S3G-I). Moreover, DNMT3B knockdown increased the mRNA and protein levels of PPARGC1A and LRBA but not ATP8A1 ( Figure 4I; Figure S4A). TET2 overexpression also promoted the protein level of PPARGC1A but reduced LRBA and ATP8A1 protein levels ( Figure 4J). Therefore, PPARGC1A may be a downstream target of DNMT3B and TET2. PPARGC1A levels in CRC were negatively associated with DNMT3B levels and positively associated with TET2 levels ( Figure S4B-E). Studies have shown that PPARGC1A mediates mitochondrial  Data are presented as mean ± standard deviation. *p < .05; **p < .01; ***p < .001. AA, advanced adenoma; CRC, colorectal carcinoma; 5mC, 5-methylcytosine; 5hmC, 5-hydroxymethylcytosine. biogenesis and energy metabolism to regulate tumourigenesis in CRC. 8,9 Our results also showed that PPARGC1A was markedly decreased in CRC, and PPARGC1A overexpression inhibited cell proliferation, invasion, and migration in HCT116 cells ( Figure 4K,L; Figure S4F-H). Moreover, PPARGC1A expression was positively correlated with activated dendritic cells, memory resting CD4 T cells, and also related to energy metabolism and mitochondrial gene expression ( Figure S4I-K; Additional File 5). In addition, compared with the AA group, the 5-mC level of PPARGC1A was markedly increased and 5-hmC level of PPARGC1A was markedly decreased in the CRC group (Figure 4 M,N). These results indicated that PPARGC1A mediated by DNMT3B and TET2 could regulate AA-CRC transformation. 10 In summary, 5mC and 5hmC showed dynamic changes in the progression of AA-CRC transformation. Mechanistically, DNMT3B knockdown and TET2 overexpression inhibited CRC progression. Finally, DNMT3B-mediated 5mC and TET2-mediated 5hmC regulated PPARGC1A expression, which could regulate the progression of AA-CRC transformation ( Figure 4O). Our results not only suggest critical roles of DNMT3B and TET2 in the AA-CRC transformation but also provide a new strategy for CRC treatment.

A C K N O W L E D G E M E N T S
The authors would like to thank Cloud-Seq Biotech Inc. (Shanghai, China) for MeDIP-seq and NewCore Biotech (Shanghai, China) for bioinformatics analysis of the data. This work was supported by the Shanghai Rising-Star Program (grant number: 21QA1409000) and Shanghai Frontier Research Base of Disease and Syndrome Biology of Inflammatory Cancer Transformation (grant number: 2021KJ03-12).

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no competing interests.